Geminiviruses are plant pathogenic, single stranded DNA viruses that cause major losses in a number of important crops throughout the world. In many areas, geminiviruses are the greatest constraint on production of important crops, including cassava, beans, cowpeas, peppers, tomatoes, and cotton. In the continental U.S., geminiviruses cause severe problems in tomatoes, peppers, melons, and beans. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes severe disease in beans (Phaseolus vulgaris) in the Western Hemisphere. In Latin America, where beans are an important food staple, BGMV infection of bean plants is the major cause of reduced bean production. BGMV also causes substantial crop loss in pole beans in Florida.
Geminiviruses replicate via a rolling circle type of mechanism (Saunders et al., Nucleic Acids Res. 19:2325-2330, 1991; Stenger, et al. Proc. Natl. Acad. Sci. USA 88:8029-8033, 1991) similar to bacteriophages such as .phi. X174. A geminivirus gene known as the rep gene encodes a complex, multifunctional protein, which acts as a rolling circle initiator protein analogous to protein A of .phi. X174. The rep gene is the only geminiviral gene necessary for viral replication (Elmer., Nucleic Acid Res. 16:7043-7060, 1988; Etessami et al., J. Gen. Virol. 72:1005-1012, 1991; Hanley-Bowdoin et al., Proc. Natl. Acad. Sci. USA 87:1446-1450, 1990).
The origin of replication (ori) of geminiviruses is characterized by a series of direct repeat sequences located between the transcription and translation start sites for the rep gene (Eagle et al., Plant Cell 6:1157-1170, 1994; Faria et al., Phytopathology 84:7043-7060, 1994; Fontes et al., J. Biol. Chem. 269:8459-8465, 1994). The Rep proteins from tomato golden mosaic virus (TGMV) and BGMV-GA (Guatemalan isolate) have been shown to bind to these direct repeats (Chapter V; Fontes et al., Plant Cell 4:597-608, 1992). The interaction between the Rep protein and the direct repeats is necessary for replication and auto-regulation of the rep gene (Eagle et al., Plant Cell 6:1157-1170, 1994; Fontes et al., Plant Cell 6:405-416, 1994). The direct repeats are generally conserved in position but vary in sequence between different phylogenetic clusters of geminiviruses. The interaction between the Rep protein and the ori is highly specific. Rep proteins from BGMV-GA and TGMV are able to interact with their cognate ori sequences but not to heterologous cri sequences (Chapter V; Fontes, et al. J. Biol. Chem. 269:8459-8465, 1994).
There exists no known natural resistance to geminiviruses. Efforts to develop geminivirus-resistant beans and tomatoes have been underway for about two decades. These breeding programs have been generally unsuccessful (Beebe., Crop Science 35:1178-1183, 1995; Morales et al., Euphytica 52:113-118, 1991). The tyl gene, which confers partial resistance to TYLCV, has been identified in wild tomato species (Nateshan et al., In Bemisia 1995: Taxonomy. Biology. Damage, Control and Management D. G. a. R. T. Mayer, Ed., pp 369-377. Intercept Ltd.; ZAMIR 1994). However, the tyl gene is not effective against other tomato- infecting geminiviruses such as ToMoV and has been difficult to introgress into commercial cultivars. A source of partial BGMV resistance, a recessive gene (bgm1) from breeding A429. has been identified for beans. The bgm1 gene has recently been incorporated into pole beans in Florida and into small red beans in Honduras. It is not known if the bgm 1 gene will confer resistance to other bean-infecting geminiviruses such as BGMV-BZ (Brazil), bean dwarf mosaic virus (BDMV), or bean calico mosaic virus.
The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of virus-derived resistance strategies (Frischmuth and Stanley, Sem. Virol. 4:329-337, 1993). Because the rep gene is the only gene necessary for geminiviral DNA replication (Hanley-Bowoin et al. Proc. Natl. Acad. Sci. USA 87:1446-1450, 1990), it is an attractive target for engineering geminivirus resistance.
What is needed in the art is a method of interfering with the function of geminiviral rep genes so as to inhibit replication of geminivirus in plants.